Maintaining unperturbed cerebral blood flow is key in the study of brain metastasis and its interactions with stress and inflammatory responses.

Blood-borne brain metastases are associated with poor prognosis, but little is known about the interplay between cerebral blood flow, surgical stress responses, and the metastatic process. The intra-carotid inoculation approach, traditionally used in animal studies, involves permanent occlusion of the common carotid artery (CCA). Herein we introduced a novel intra-carotid inoculation approach that avoids CCA ligation, namely - assisted external carotid artery inoculation (aECAi) - and compared it to the traditional approach in C57/BL6 mice, assessing cerebral blood flow; particle distribution; blood-brain barrier (BBB) integrity; stress, inflammatory and immune responses; and brain tumor retention and growth. Doppler flowmetry and two-photon imaging confirmed that only in the traditional approach regional and capillary cerebral blood flux were significantly reduced. Corticosterone and plasma IL-6 levels were higher in the traditional approach, splenic numbers of NK, CD3+, granulocytes, and dendritic cells were lower, and many of these indices were more profoundly affected by surgical stress in the traditional approach. BBB integrity was unaffected. Administration of spherical beads indicated that CCA ligation significantly limited brain distribution of injected particles, and inoculation of D122-LLC syngeneic tumor cells resulted in 10-fold lower brain tumor-cell retention in the traditional approach. Last, while most of the injected tumor cells were arrested in extra-cranial head areas, our method improved targeting of brain-tissue by 7-fold. This head versus brain distribution difference, commonly overlooked, cannot be detected using in vivo bioluminescent imaging. Overall, it is crucial to maintain unperturbed cerebral blood flow while studying brain metastasis and interactions with stress and inflammatory responses.

Alginate-coated magnetic nanoparticles for noninvasive MRI of extracellular calcium.

Nanoparticles (NPs) have great potential to increase the diagnostic capacity of many imaging modalities. MRI is currently regarded as the method of choice for the imaging of deep tissues, and metal ions, such as calcium ions (Ca(2+)), are essential ingredients for life. Despite the tremendous importance of Ca(2+) for the well-being of living systems, the noninvasive determination of the changes in Ca(2+) levels in general, and extracellular Ca(2+) levels in particular, in deep tissues remains a challenge. Here, we describe the preparation and contrast mechanism of a flexible easy to prepare and selective superparamagnetic iron oxide (SPIO) NPs for the noninvasive determination of changes in extracellular Ca(2+) levels using conventional MRI. We show that SPIO NPs coated with monodisperse and purified alginate, having a specific molecular weight, provide a tool to selectively determine Ca(2+) concentrations in the range of 250 µm to 2.5 mm, even in the presence of competitive ions. The alginate-coated magnetic NPs (MNPs) aggregate in the presence of Ca(2+) , which, in turn, affects the T2 relaxation of the water protons in their vicinity. The new alginate-coated SPIO NP formulations, which have no effect on cell viability for 24 h, allow the detection of Ca(2+) levels secreted from ischemic cell cultures and the qualitative examination of the change in extracellular Ca(2+) levels in vivo. These results demonstrate that alginate-coated MNPs can be used, at least qualitatively, as a platform for the noninvasive MRI determination of extracellular Ca(2+) levels in myriad in vitro and in vivo biomedical applications.

Preconditioning to mild oxidative stress mediates astroglial neuroprotection in an IL-10-dependent manner.

Oxidative stress plays an important role in the pathogenesis of various brain insults, including stroke. Astroglia are the main glial cells that play a fundamental role in maintaining the homeostasis of the CNS. They are important for protection from injury and aid the brain in functional recovery after injuries. It has been shown that the brain can be prepared to withstand an oxidative stress insult by a process known as preconditioning. We used primary astroglial cell culture to investigate whether preconditioning to mild oxidative stress and glucose deprivation (OSGD) can increase both astroglia survival and neuroprotective features. We found that preconditioning astroglia to mild OSGD increases astroglial survival of a second insult through activation of the NF-E2-related factor-2 (Nrf-2) pathway. Moreover, we found that Nrf-2 is highly expressed in adult brain astroglia and that preconditioning to OSGD in vivo, such as in a murine model of ischemic stroke, leads to a significant increase in astroglial Nrf-2 expression. Furthermore, we discovered an increase in neuroprotection, as measured by increased neuronal cell survival, following OSGD in the presence of medium from astroglia exposed to a mild OSGD condition. Interestingly, we discovered a significant increase in astroglial secretion of the anti-inflammatory cytokine IL-10 vs. the pro-inflammatory cytokine IL-1ß in mild vs. severe oxidative stress, respectively. We demonstrated that preconditioning astroglia to mild oxidative stress increases neuroprotection in an IL-10-dependent manner. By using tert-butylhydroquinone (tBHQ), a known specific activator of Nrf-2, we found that Nrf-2 can enhance IL-10 expression. Further studies of Nrf-2-mediated cellular pathways in astroglia through IL-10 may provide useful insights into the development of therapeutic interventions following oxidative stress insults such as ischemic stroke.

A role for vascular deficiency in retinal pathology in a mouse model of ataxia-telangiectasia.

Ataxia-telangiectasia is a multifaceted syndrome caused by null mutations in the ATM gene, which encodes the protein kinase ATM, a key participant in the DNA damage response. Retinal neurons are highly susceptible to DNA damage because they are terminally differentiated and have the highest metabolic activity in the central nervous system. In this study, we characterized the retina in young and aged Atm-deficient mice (Atm(-/-)). At 2 months of age, angiography revealed faint retinal vasculature in Atm(-/-) animals relative to wild-type controls. This finding was accompanied by increased expression of vascular endothelial growth factor protein and mRNA. Fibrinogen, generally absent from wild-type retinal tissue, was evident in Atm(-/-) retinas, whereas mRNA of the tight junction protein occludin was significantly decreased. Immunohistochemistry labeling for occludin in 6-month-old mice showed that this decrease persists in advanced stages of the disease. Concurrently, we noticed vascular leakage in Atm(-/-) retinas. Labeling for glial fibrillary acidic protein demonstrated morphological alterations in glial cells in Atm(-/-) retinas. Electroretinographic examination revealed amplitude aberrations in 2-month-old Atm(-/-) mice, which progressed to significant functional deficits in the older mice. These results suggest that impaired vascularization and astrocyte-endothelial cell interactions in the central nervous system play an important role in the etiology of ataxia-telangiectasia and that vascular abnormalities may underlie or aggravate neurodegeneration.

γ-Secretase component presenilin is important for microglia ß-amyloid clearance.

OBJECTIVE: The cleavage of amyloid precursor protein by γ-secretase is an important aspect of the pathogenesis of Alzheimer's disease. γ-Secretase also cleaves other membrane proteins (eg, Notch), which control cell development and homeostasis. Presenilin 1 and 2 are considered important determinants of the γ-secretase catalytic site. Our aim was to investigate whether γ-secretase can be important for microglial phagocytosis of Alzheimer's disease ß-amyloid. METHODS: We investigated the role of γ-secretase in microglia activity toward ß-amyloid phagocytosis in cell culture using γ-secretase inhibitors and small hairpin RNA and presenilin-deficient mice. RESULTS: We found that γ-secretase inhibitors impair microglial activity as measured in gene expression, protein levels, and migration ability, which resulted in a reduction of soluble ß-amyloid phagocytosis. Moreover, microglia deficient in presenilin 1 and 2 showed impairment in phagocytosis of soluble ß-amyloid. Dysfunction in the γ-secretase catalytic site led to an impairment in clearing insoluble ß-amyloid from brain sections taken from an Alzheimer's disease mouse model when compared to microglia from wild-type mice. INTERPRETATION: We suggest for the first time, a dual role for γ-secretase in Alzheimer's disease. One role is the cleavage of the amyloid precursor protein for pathologic ß-amyloid production and the other is to regulate microglia activity that is important for clearing neurotoxic ß-amyloid deposits. Further studies of γ-secretase-mediated cellular pathways in microglia may provide useful insights into the development of Alzheimer's disease and other neurodegenerative diseases, providing future avenues for therapeutic intervention.

The P4 promoter of the parvovirus minute virus of mice is developmentally regulated in transgenic P4-LacZ mice.

Activation of the minute virus of mice (MVM) P4 promoter is a key step in the life cycle of the virus and is completely dependent on host transcription factors. Since transcription-factor composition varies widely in different cell types, there is the possibility that only some cell types in the host organism have the capacity to initiate expression from the P4 promoter and therefore that the promoter may be a factor in determining the tropism of MVM. In this study, the ability of various cell types to activate P4, independent of the other virus-host interactions, was examined in transgenic mouse lines bearing a beta-galactosidase reporter sequence driven by the P4 promoter. It was found that lacZ was expressed during embryogenesis and in the adult in a cell-type-specific and differentiation-dependent pattern. The data are consistent with cell-type and stage-specific activation of the P4 promoter having a role in determining the host cell-type range of MVM. The ability of some parvoviruses to replicate in, and kill oncogenically transformed cells, and to destroy induced tumors in laboratory animals is the basis of recent approaches to use MVM-based vectors in cancer gene therapy. Since these vectors rely on the activation of the P4 promoter by the target tissues, understanding the promoter dependence on cell-type and differentiation status is important for their design and potential use.